Project Summary/Abstract There is a fundamental gap in the understanding of how tobacco flavorants alter nicotine addiction. Since electronic nicotine delivery systems (ENDS) offer a multitude of flavors, there is a critical need to under- stand how these flavors alter nicotine addiction. Until this knowledge gap is closed, we face the risk of in- creased smoking initiation, decreased cessation, and a cumulative effect of a growing population of lifelong smokers in America. Our overall goal is to identify a biomarker to test which flavorants enhance nicotine addic- tion and abuse liability. This is a major priority of the FDA-CTP. We hypothesize that ?4?6?2 nicotinic acetylcholine receptor (nAChR) upregulation is a suitable bi- omarker for identifying flavorants that enhance nicotine addiction. The rationale behind this comes from the applicant?s previous findings that only ?4?6?2 nAChRs are upregulated by concentrations of nicotine that are sufficient to elicit reward-related behavior in animal models. Therefore, ?4?6?2 nAChRs present a potential biomarker that is more sensitive when compared to other nAChR subtypes. Furthermore, the applicant has shown that an enhancement in nicotine reward by menthol, the most popular tobacco flavorant, is accompa- nied by enhancements in ?4?6?2 nAChR upregulation. This strong preliminary data suggests that these meth- ods can be utilized to develop ?4?6?2 nAChR upregulation as a novel biomarker to examine other flavorants for their role in enhancing nicotine addiction and abuse liability. This novel biomarker will be validated by exam- ining two chemicals used in green apple and fruit flavorants (farnesol and farnesene) in three specific aims: 1) characterization of flavorants? effect (farnesol and farnesene) on nicotine reward-related behavior; 2) character- ization of flavorants? effect on nicotine-induced ?4?6?2 nAChR upregulation; and 3) characterization of flavor- ants? effect on nicotine-induced changes in dopamine neuron excitability. In the first aim, a mouse conditioned place preference assay identical to one used to report menthol-induced enhancements in nicotine reward will determine if the flavorants farnesene and farnesol enhance nicotine reward. These assays will be supplement- ed with mouse self-administration assays utilizing e-VapeTM technology. The second aim will use the brains from Aim 1 mice to examine ?4?6?2 nAChR upregulation and provide a direct correlation between enhance- ments in nicotine reward and ?4?6?2 nAChR upregulation. This will be a critical feature to validate our bi- omarker assay. Aim 3 will provide validation measures that will supplement upregulation using electrophysio- logical methods to examine changes in dopamine neuron excitability. This approach is innovative, in the appli- cant's opinion, because it establishes a direct correlation between nicotine reward and nAChR upregulation in an in vivo model. This is complemented by the use of a novel mouse expressing ?4-mCherry and ?6-GFP nA- ChR subunits that allow analysis of upregulation without the use of antibodies. The proposed research is signif- icant, because it presents a novel biomarker method to identify tobacco flavorants that pose a risk to nicotine addiction and abuse liability. This would contribute significantly to several of the FDA-CTP research priorities.